Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally Post, Secunderabad, Hyderabad, Telangana - 500014
Vericose vein is condition that causes veins to bulge, swelling, twisting veins just below the skin’s surface most often in the leg and feet. They are also offered in topical treatments that provide relief from associated symptoms such as pain, swelling, and inflammation, but can be inconvenient to frequent application. In this study we developed a novel herbal transdermal patch for varicose vein management using Curcuma longa (turmeric) and Centella asiatica extracts. Anti-inflammatory and wound healing properties are attributed to curcuma longa and collagen synthesis with antioxidant activity to Centella asiatica. The extracted plant materials were authenticated using great care before extraction using Soxhlet and maceration yields 37% w/w and 23.12% w/w respectively. Curcuminoids in Curcuma longa extracts and triterpenoids in Centella asiatica extracts were confirmed by thin layer chromatography. Since both extracts have poor lipid solubility, transdermal patches were selected to increase skin penetration. Three HPMC based patch formulations (F1, F2, F3) were developed and these were subsequently investigated for stability and in vivo in rat. F1 and F2 were loaded with HPMC 50, and with F3 a mixture of HPMC 50 and HPMC 15 was used. Pharmaceutical characterization of formulations was performed by organoleptic evaluation, pH determination, folding endurance, thickness, drug uniformity, drug release, in vitro antioxidant activity, and in vitro anti inflammation activity. According to all parameters, HPMC polymer combination in Batch F3 had optimal performance, holding and releasing both extracts in a controlled manner. Further stability studies also demonstrated the suitability of F3 formulation. Hence, the F3 Formula is completed for the herbal transdermal patch for varicose vein management.
Varicose veins are a condition where veins, typically in the legs, become permanently enlarged, twisted, and painful. This condition is often due to improperly functioning valves in the vein or weakness in the vein walls [1]. The causes of varicose veins are multifactorial and include increased intravenous pressure from prolonged standing, increased intra-abdominal pressure due to factors like tumors, pregnancy, obesity, or chronic constipation, as well as familial and congenital factors. Secondary vascularization from deep venous thrombosis or, less commonly, arteriovenous shunting, also contributes to their development [2]. Recent research highlights shear forces and inflammation as significant factors in venous disease etiology [3]. The fundamental pathophysiology involves valve reflux in the veins, where failed or incompetent valves cause blood to flow in reverse. This reverse flow increases pressure on the local venous system, causing affected veins to elongate and become tortuous. The specific cause of valvular dysfunction is generally thought to be the loss of elasticity in the vein walls, leading to valve leaflets that no longer fit together properly [4].
The prevalence of varicose veins varies by region, affecting approximately 2% to 73% of the global population [5]. It is estimated that about 33% of individuals aged 18 to 64 years are affected by varicose veins, which contributes significantly to healthcare costs. In Western countries, varicose veins are found in 10% to 15% of males and 20% to 25% of females [5].
Treatment options for varicose veins encompass conservative management, external laser treatment, injection sclerotherapy, endovenous interventions, and surgery. The choice of treatment is often guided by patient preference but also depends on factors such as symptom severity, cost, potential for complications, available medical resources, insurance coverage, physician expertise, the presence of deep venous insufficiency, and the characteristics of the affected veins [6].
Transdermal patches represent an innovative and effective approach for delivering medications directly through the skin to treat varicose veins. This method offers several advantages over traditional oral or injectable medications, including improved patient compliance, controlled drug release, and reduced systemic side effects. Transdermal patches consist of a drug reservoir or matrix that contains the active pharmaceutical ingredient. When applied to the skin, the drug diffuses through the stratum corneum (the outermost layer of the skin) and enters the systemic circulation, or it can act locally to relieve symptoms associated with varicose veins.
In present study, we aimed to prepare and evaluate transdermal patch prepared by using Curcuma longa and Centella asiatica extracts for management of Vericose vein. Curcuma longa is well known anti-inflammatory herb while Centella asiatica is found to be effective in strengthening the blood capillary wall and also it promotes synthesis of collagen. In this study, we prepared C. longa and C. asiatica extracts by using ethanol as solvent. Transdermal patch was prepared by using polymers like HPMC 50, HPMC 15, plasticizers like polyethylene glycol, propylin glycol etc. The prepared formulation was evaluated by the physicochemical and biological parameters to make sure that formulation is successfully developed.
MATERIALS AND METHODS
Plant Material
Based on extensive literature review and existing research work, Centella asiatica and Curcuma longa were selected for transdermal patch formulation. Dried powders of Centella asiatica and Curcuma longa were procured from authorized herbal drugs supplier. The procured plant materials were authenticated by the chemical tests for presence of the desired phytochemicals before using for the formulation.
Chemicals
Hydroxy propyl methyl cellulose 15, Hydroxy propyl methyl cellulose 50, Ethyl alcohol, Propylene glycol, Polyethylene Glycol 400, distilled water, 2,2 diphenyl-1-picrylhydrazyl (DPPH) procured from CDH Chemicals, New Delhi
Preliminary Phytochemical evaluation of plant material
Preliminary phytochemical investigation of procured dried powders of asiatica and Curcuma longa was done by conducting drug specific chemical tests. Following chemical tests were used:
Table 1. Chemical tests for identification of Curcuma longa and Centella asiatica
|
Sr. No |
Name of Plant extract |
Chemical test |
Ref |
|
1 |
Curcuma longa extract |
Treatment with sulphuric acid gives red color |
[7] |
|
Treatment with alkali solution gives red to violet color |
|||
|
.With acetic anhydride and concentrated sulphuric acid gives violet colour. Under UV light this colour is seen as an intense red fluorescence. |
|||
|
with borax solution gives .a green color |
|||
|
With boric acid gives reddish-brown color which, on addition of alkalies, changes to greenish-blue |
|||
|
2 |
Centella asiatica extract |
a) Foam test: - Shake the drug extract or dry powder vigorously with water b) Shinoda test: - To dry powder or extract, add 5 ml 95% ethanol/t-butyl alcohol, few drops conc.HCL and 0.5 g magnesium turnings. c) Salkowski reaction: - To 2 ml extract, add 2 ml chloroform and 2ml conc.H2SO4. Shake well. d) Borntrager’s test :1gm of drug sample + 5-10 ml of dilute HCl + 10 min. boil on waterbath and filter + extract of filterate with CCl4 or benzene + equal amount of ammonia solution to filterate + shake ? appearance of pink to red colour ? indicate presence of anthraquinone moiety. e) Libermann Burchard test : Alcoholic extract of drug ? evaporated ? dry ?extracted with CHCl3 + few drops of acetic anhydride + conc.salphuric acid ( from the side wall of test tube) ? appearance of violet ring ? blue colour ? presence of sterol group in drug. |
Extract Preparation
Preparation of Curcuma longa Extract
Ethanolic extract of curcuma longa was prepared by the Soxhlet extraction technique. 100 gms of curcuma longa powder was transferred in the Soxhlet apparatus and extracted for 6 hours with 350 ml of 95% ethanol at 40 – 50 ºC temperature. After complete extraction, the extract was removed from the round bottom flask and concentrated at low temperature by evaporating excess of ethanol [8].
Preparation of Centella asiatica extract
Extraction of Centella asiatica was performed by maceration technique. In this method, 100 gm of Centella asiatica powder was added to 300 ml of 95% of ethanol in a 500 ml beaker. The mixture was kept for maceration with occasional stirring for 24 hrs at room temperature. After 24 hours, the mixture was filtered and extract was collected in conical flask. Again 300 ml of 95% of ethanol was added to the mark and kept for maceration for 24 hrs. The same process was repeated three times to ensure the complete extraction. All filtrates were collected together and concentrated to obtain percentage yield of extract [9]
Qualitative analysis of extracts by TLC
Thin layer chromatography of extracts of curcuma longa, Centella asiatica was performed using precoated Silica Gel G F254 to ensure the presence of desired phytochemicals in the extracts. Sample solutions for spot application on plate were prepared by dissolving 1 mg of drug as well as reference standards in 5 ml methanol in separate test tubes. Purified Curcumin and asiaticosides used as reference standards. The mobile phases used for TLC analysis are as follows:
Table 2. Mobile phases for TLC analysis
|
Sr. No |
Name of Extract |
Mobile Phase |
|
1 |
Curcuma longa ethanolic extract |
Chloroform: methanol (97:3V/V) [10] |
|
2 |
Centella asiatica alcoholic extract |
Chloroform: glacial acetic acid: Glacial Acetic Acid: methanol: water. (6:3.2:1.2:8) [9] |
Preparation of Transdermal patch formulation
Transdermal patch formulation was optimized by preparing three different batches F1, F2, F3 by varying the quantities of ingredients (as shown in table 3).
Table 3. Batchwise preparation of herbal transdermal patch for optimization of formulation
|
Batch Code |
Polymers |
Chemicals |
Excipients |
Extracts |
|
F1 |
HPMC 15 cps |
3 gm of HPMC 15 + 40 ml of distilled water qs |
Propylene Glycol 15%+ PEG 400 30% |
Curcuma longa extract 5%, Centella asiatica extract 5 % |
|
F2 |
HPMC 50 cps |
3 gm of HPMC 50+ 40 ml of distilled water qs |
Propylene Glycol 15%+ PEG 400 30% |
Curcuma longa extract 5%, Centella asiatica extract 5 % |
|
F3 |
HPMC 15 cps+HPMC 50 cps |
1.5 gm of HPMC 15 +1.5 gm of HPMC 50 +40 ml of distilled water |
Propylene Glycol 15%+ PEG 400 30% |
Curcuma longa extract 5%, Centella asiatica extract 5 % |
Transdermal patches with selected plant extracts were prepared by solvent casting method using Petri plates. Polymers HPMC 15, HPMC 50 and their combination were soaked in distilled water in separate beakers respectively for 24 hours at room temperature. After soaking, polymers were homogenized by using magnetic stirrer; the method used for transdermal patch formulation is as follows:
Initially polymers used in F1, F2 and F3 were kept for homogenization on magnetic stirrer for 1 hr at 100 rpm speed. Plant extracts of curcuma longa and Centella asiatica (500 mg each) were dissolved in 5 ml ethanol completely. Extracts were homogenized on magnetic stirrer for 30 minutes at 100 rpm. Extract mixture was added to the polymer with continuous stirring on magnetic stirrer. Following addition of extracts, plasticizers PEG 400 and propylene glycol were added to the mixture and stirred well on magnetic stirrer for 1 hour. Each formulation was made up to 50 ml quantity using distilled water during homogenization process. After homogenization, the mixture was poured in petri plates in such a quantity that patch will become very thin [11].
Characterization of Formulation
Following parameters were evaluated to confirm the therapeutic efficacy of prepared formulation:
a) Organoleptic Evaluation
All the trial batches of transdermal patch formulations were tested for the color, odor, texture and appearance [12].
b) Determination of pH
Digital pH meter was used for the determination of the pH of F1, F2, F3 formulations of transdermal patch. For PH measurement, Transdermal patch was solublized in small amount of distilled water. PH detection rod was immersed in the solution and PH was recorded of each batch [12].
c) Thickness of formulation
The thickness of each formulation (F1, F2, and F3) was measured at five different locations (centre and four corners) using vernier caper and it was averaged to determine the mean thickness in mm [12].
d) Folding Endurance
Folding endurance of the patch formulation of each batch was determined by repeatedly folding one patch formulation at the same place till it broke [12].
e) Weight variation study
Ten samples of patch of each batch were weighed individually in a digital balance and the mean weights were calculated. It is useful to ensure that a film contains the proper number of excipients and API [12].
f) ) Study of drug excipient compatibility by FTIR
FTIR spectroscopy was conducted to examine the interactions between the extracts and excipients. The analysis utilized a Shimadzu FTIR Spectrophotometer, with the extracts serving as controls for comparative assessment of interactions [12].
g) Drug Uniformity Test
A transdermal patch (2x2 cm) was dissolved in 100 ml of methanol and subjected to continuous shaking for 24 hours. The resulting solution was then sonicated for 15 minutes and filtered. The concentration of the drug in the filtered solution was determined spectrophotometrically at a wavelength of 292 nm [12].
h) Invitro Drug Release studies
In vitro drug release studies were conducted using a Franz diffusion cell system. The receptor compartment, with a 60 ml capacity, was filled with pH 7.4 phosphate buffer and maintained at 37±0.5°C to simulate normal human body temperature. A cellulose acetate membrane with a 0.45 ?m pore size separated the donor and receptor compartments. Prepared transdermal patches were mounted on the membrane and sealed with aluminum foil. The receptor compartment solution was continuously stirred at 50 rpm using magnetic beads, following a method described by Simon et al. Samples were withdrawn at predetermined intervals, analyzed spectrophotometrically for drug content, and replaced with an equal volume of fresh phosphate buffer. Care was taken during manual sampling to avoid introducing air bubbles into the receptor compartment [12].
g) Invitro drug efficacy studies
The therapeutic efficiency of prepared herbal transdermal patch was analyzed by using invitro assays of free radical scavenging activity by DPPH and invitro anti-inflammatory assay since free radicals and chronic inflammation are one of the well identified causative factors of psoriasis development.
i) Antioxidant activity by DPPH assay
Antioxidant activity was evaluated using the DPPH free radical scavenging assay, with gallic acid serving as the reference standard. A 1 mg/ml stock solution of gallic acid was prepared in ethanol and serially diluted to concentrations ranging from 10-50 µg/ml. Similar serial dilutions (10-50 µg/ml) were prepared for each batch of transdermal patches. A DPPH working solution was prepared by dissolving 10.83 mg of DPPH in ethanol and adjusting the final volume to 100 ml. Absorbance readings were taken at 520 nm, using the solvent (ethanol) as a blank and a DPPH solution without the transdermal patch formulation as a control [13].
Following formula used to calculate % free radical scavenging potential of prepared Transdermal patch: % Inhibition of free radicals = Absorbance of blank – Absorbance of test/ Absorbance of Blank
ii) Invitro anti-inflammatory assay
The anti-inflammatory potential of different emulgel formulations (F1-F3) was evaluated in vitro using a red blood cell membrane stabilization assay. Sheep red blood cells (50 µl) were exposed to hypotonic solutions containing varying concentrations (12.5, 25, 50, and 100 ppm) of the transdermal patch formulations. A control group without the test formulations was also included. The isotonic solution consisted of 154 mM NaCl in 10 mM sodium phosphate buffer (pH 7.4). After a 10-minute incubation at room temperature, the samples were centrifuged at 5000 rpm for 5 minutes. The absorbance of the supernatant was measured at 540 nm using a UV spectrophotometer. Diclofenac sodium (200 µg/ml) was used as a reference standard. The % inhibition of red blood cell lysis was calculated by following formula [14]:
% Red blood Cell membrane stability = 100 × [1 – OD2 – OD1/OD3 – OD1]
Here, OD1 represents the test sample in an isotonic solution, OD2 denotes the test sample in a hypotonic solution, and OD3 serves as the control sample in a hypotonic solution.
h) Stability Study
Stability testing was performed in alignment with the International Council for Harmonization (ICH) guidelines. The finalized Transdermal patch formulation batch based on the above assessments underwent accelerated stability testing over a 3-month period under controlled conditions of 40 ± 2?C temperature and 75 ± 5% relative humidity.
i) Statistical analysis
All values are expressed as Mean ± SD. All data was analysed statistically by Single factor ANOVA (Analysis of Variance) by using graph pad prism Ver 5.1.0. The p value less than 0.05 (p < 0>
Phytochemical evaluation of plant material outcomes
Selected plant materials Centella asiatica and Curcuma longa were examined by conducting phytochemicals specific chemical tests. Outcomes of the chemical tests are as follows (Table 4):
Table 4. Outcomes of preliminary phytochemical identification chemical tests
|
Sr. No |
Name of Plant extract |
Chemical test |
Inference |
|
1 |
Curcuma longa extract |
Treatment with sulphuric acid gives red color |
Curcuminoids present |
|
Treatment with alkali solution gives red to violet color |
Curcuminoids present |
||
|
.With acetic anhydride and concentrated sulphuric acid gives violet colour. Under UV light this colour is seen as an intense red fluorescence. |
Curcuminoids present |
||
|
with borax solution gives .a green color |
Curcuminoids present |
||
|
With boric acid gives reddish-brown color which, on addition of alkalies, changes to greenish-blue |
Curcuminoids present |
||
|
2 |
Centella asiatica extract |
a) Foam test: - Shake the drug extract or dry powder vigorously with water b) Shinoda test: - To dry powder or extract, add 5 ml 95% ethanol/t-butyl alcohol, few drops conc.HCL and 0.5 g magnesium turnings. c) Salkowski reaction: - To 2 ml extract, add 2 ml chloroform and 2ml conc.H2SO4. Shake well. d) Borntrager’s test :1gm of drug sample + 5-10 ml of dilute HCl + 10 min. boil on water bath and filter + extract of filterate with CCl4 or benzene + equal amount of ammonia solution to filterate + shake ? appearance of pink to red colour ? indicate presence of anthraquinone moiety. e) Libermann burchard test : Alcoholic extract of drug ? evaporated ? dry ?extracted with CHCl3 + few drops of acetic anhydride + conc.salphuric acid ( from the side wall of test tube) ? appearance of violet ring ? blue colour ? presence of sterol group in drug. |
Saponins present Glycosidal flavanoids present, Steroids present, Glycosidal saponins present |
Hence, from above chemical tests, procured plant materials found authenticate and free from adulterants for further use in formulation
Extract preparation outcomes
Extracts of Curcuma longa was prepared by soxhlet extraction method by using 95% alcohol as a solvent. Ethanol selected as a solvent because curcuminoids are soluble in ethanol and insoluble in water. The percentage yield of the extract was found to be 37 %. Centella asiatica is rich source of saponin glycosides compounds which are soluble in solvents like ethanol hence; it was used as solvent for extraction. The % yield of the Centella extract was found to be 23.12 %.
Qualitative analysis of extracts by TLC
Qualitative analysis of extracts for presence of desired phytochemicals was performed by Thin Layer Chromatography. In this method Rf value of reference standard is compared with the spots obtained from extract. For TLC analysis of curcuma longa and Centella asiatica extracts, curcumin and asiaticosides were used as reference standard The outcomes of the TLC analysis are as follows (Table 5)
Table 5. The outcomes of the TLC analysis
|
Sr. No |
Name of Extract |
Mobile Phase |
Rf values obtained |
|
1 |
Curcuma longa ethanolic extract |
Chloroform: methanol (97:3V/V) |
curcumin extract = 0.38 Std. Curcumin = 0.39 |
|
2 |
Centella asiatica alcoholic extract |
Chloroform: glacial acetic acid: Glacial Acetic Acid: methanol: water. (6:3.2:1.2:8) |
Centella extract = 0.34 Std. Asiaticoside = 0.35 |
The Rf values of curcumin and asiaticosides in extracts of Curcuma longa and Centella asiatica was found to be 0.38 and 0.34 respectively. These values found similar to the Rf values of respective reference standards. Hence the presence of phytoconstituents curcumin, and asiaticosides confirmed in extracts
4.4 Characterization outcomes of transdermal Formulation
The characterization of transdermal patch formulations is crucial for assessment of their physical, chemical, and functional properties, which affects their stability and efficacy. The characterization involved analyzing parameters like organoleptic evaluation, pH determination, thickness, folding endurance, moisture content determination, drug content uniformity test, invitro drug release study and invitro efficacy studies like antioxidant activity and anti-inflammatory activity to ensure the formulation is appropriate for use. The outcomes of the characterization of transdermal patch formulation are as follows:
a) Organoleptic parameters evaluation
All the trial batches of Transdermal patch formulations were tested for the color, odor, texture and appearance. The outcomes of the test are discussed in the table as follows (Table 6):
Table 6. The outcomes of the organoleptic test
|
Sr. No |
Parameters |
F1 |
F2 |
F3 |
|
1 |
Color |
Yellowish |
Yellowish |
Yellowish |
|
2 |
Odor |
Characteristic |
Characteristic |
pleasant |
|
3 |
Texture |
Slightly rough |
smooth |
Smooth |
|
4 |
Appearance |
solid |
solid |
solid |
b) Determination of pH
Determination of pH of the transdermal patch is a crucial parameter to investigate the suitability of patch with the skin pH. The outcomes of test are as follows:
Table 7. Outcomes of pH determination
|
Parameters |
F1 |
F2 |
F3 |
|
pH |
6 ± 0.10 |
6.2±0.66 |
6.5±0.12 |
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
Topical formulations pH should be compatible with human skin unless it causes skin irritation and reduces patient acceptability. Ideal pH of human skin is 6.5 – 6.8. Hence, the pH of the formulation batches F3 found to be in the range of 6.6 – 6.8 compatible with the human skin.
c) Thickness of formulation
Evaluation of thickness of transdermal patch is an important aspect as it directly affects the drug release and efficacy of the transdermal patch. The thickness of each formulation (F1, F2, and F3) was measured at five different locations (centre and four corners) using vernier caliper and it was averaged to determine the mean thickness in mm. The outcomes of these evaluation parameters are as follows:
Table 8. Outcomes of thickness determination of herbal transdermal patches
|
Sr. No |
Thickness of F1 |
Thickness of F2 |
Thickness of F3 |
|
1 |
0.34±0.02 |
0.31±0.42 |
0.20±0.54 |
|
2 |
0. 34±0.23 |
0. 33±0.25 |
0.20±0.65 |
|
3 |
0. 35±0.33 |
0. 31±0.44 |
0.20±0.78 |
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
Out of F1, F2, F3 batch formulation, F3 shown appropriate thickness as compared to the remaining two batches. Hence from this study, it is conclude that F3 batch thickness is appropriate as per the formulation requirement.
d) Folding Endurance
Folding endurance of the patch formulation indicates flexibility of the patch. The patch of each batch was determined by repeatedly folding one patch formulation at the same place till it broke. The outcomes of folding endurance are as follows (Table 9):
Table 9. The outcomes of folding endurance test of Herbal transdermal patch
|
Sr. No |
Folding endurance of F1 |
Folding endurance of F2 |
Folding endurance of F3 |
|
1 |
101 |
56 |
117 |
|
2 |
100 |
56 |
115 |
|
3 |
100 |
59 |
110 |
In terms of mean ±SD, folding endurance of batch F1, F2 and F3 found to be 100.3 ± 4.96, 57 ± 2.96 and 114 ± 4.70 respectively. Among all these batches, F3 batch has shown good folding endurance.
e) Weight variation study
five samples of patch 2 × 2 of each batch were weighed individually in a digital balance and the mean weights were calculated. It is useful to ensure that a film contains the proper number of excipients and API. The outcomes of the study are as follows:
Table 10. Weight variation study outcomes of herbal transdermal patches
|
Sr. No |
Weight of F1 (mg) |
Weight of F2 (mg) |
Weight of F3 (mg) |
|
1 |
0.124 |
0.123 |
0.124 |
|
2 |
0.125 |
0.124 |
0.121 |
|
3 |
0.123 |
0.122 |
0.121 |
|
4 |
0.126 |
0.124 |
0.122 |
|
5 |
0.124 |
0.121 |
0.124 |
No significant weight variation was found in all three batches. Hence all three batches passed the test
f) Study of drug excipient compatibility by FTIR
FTIR spectroscopy was conducted to examine the interactions between the extracts and excipients. The analysis utilized a Shimadzu FTIR Spectrophotometer, with the extracts serving as controls for comparative assessment of interactions.
|
|
|
||
|
Figure 1 FTIR spectra of Curcumin extract |
Figure 2 FTIR spectra of F3 transdermal patch |
||
|
Table 11. FTIR analysis of curcuma extract and transdermal patch F3 |
|||
|
Sr. No |
Functinal groups of curcumin |
Frequency in cm-1 of curcumin extract |
Frequency in cm-1 of F3 |
|
1 |
C=O caobonyl [satureted aliphatic] |
1743.72 |
1743.72 |
|
2 |
CH aliphatic sterchning |
2978.22 |
2985.94 |
|
3 |
CH aromatic srechning |
3364.00 |
- |
|
4 |
C=C benzene ring |
1612.56 |
1651.14 |
|
5 |
Hydroxy group |
3526.03 |
3541.46 |
|
6 |
Aldehede stechning |
2600.00 |
2947.36 |
|
7 |
C and H bond stechning |
2978.22 |
2947.36 |
|
|
|
||
|
Figure 3 FTIR spectra of Centella extract |
Figure 4 FTIR spectra of F3 transdermal patch |
||
|
Table 12. FTIR analysis of Centella extract and transdermal patch F3 |
|||
|
Sr. No |
Functional groups of Centella |
Frequency in cm-1 of Centella extract |
Frequency in cm-1 of F3 |
|
1 |
CH aromatic srechning |
2955.07 |
2985.00 |
|
3 |
C=O caobonyl [satureted aliphatic] |
1705.15 |
1743.72 |
|
4 |
C=C benzene ring |
1651.14 |
1651.14 |
|
5 |
Hydroxy group |
3570.60 |
3541.48 |
|
6 |
O-H bending |
1165.05 |
1165.00 |
From above study, it is observed that, no significant drug excipients interactions present in F3 formulation.
g) Drug Uniformity Test
Drug uniformity test is important parameter to ensure drug is uniformly blended with polymer so that dosing will be accurate. The outcomes of drug uniformity test are as follows (Table 13):
Table 13. Outcomes of drug uniformity test of herbal transdermal formulations
|
Sr. No |
% drug content F1 |
% drug content F2 |
% drug content F3 |
|
1 |
98.23 |
96.45 |
99.01 |
|
2 |
95.11 |
97.94 |
98.21 |
|
3 |
97.23 |
98.17 |
99.87 |
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
n terms of mean ±SD, The % drug release of batch F1, F2 and F3 was found to be 96.85%, 97.52% and 99.03 % respectively. Thus based on above outcomes, it is observed that, drug uniformity is more in F3 formulation as compared to other formulations.
h) Invitro Drug Release studies
In vitro drug release studies were conducted using a Franz diffusion cell system. The receptor compartment, with a 60 ml capacity, was filled with pH 7.4 phosphate buffer and maintained at 37±0.5°C to simulate normal human body temperature. A cellulose acetate membrane with a 0.45 ?m pore size separated the donor and receptor compartments. Prepared transdermal patches were mounted on the membrane and sealed with aluminum foil. The receptor compartment solution was continuously stirred at 50 rpm using magnetic beads, following a method described by Simon et al. Samples were withdrawn at predetermined intervals, analyzed spectrophotometrically for drug content, and replaced with an equal volume of fresh phosphate buffer. The UV wavelength of curcumin and Asiaticosides found to be 430 and 210 nm respectively. % drug release from transdermal patch was determined by analyzing the samples withdrawn at time intervals of 0, 50, 100, 150, 200 and 250 minutes at 430 and 210 nm respectively to find out presence of phytoconstituents in each sample. The % drug release of phytoconstituents like curcumin and asiaticosides from F1, F2 and F3 are as follows (Table 14):
Table 14 outcomes of % drug release of phytoconstituents curcumin and asiaticosides from F1, F2 and F3
|
Formulation |
% drug release at 0 min |
% drug release at 50 min |
% drug release at 100 min |
% drug release at 150 min |
% drug release at 200 min |
% drug release at 250 min |
|
|
F1 |
Curcumin |
0 |
2.0± 0.12 |
7.23± 0.22 |
13.21± 1.24 |
26.23± 0.67 |
40.19± 0.33 |
|
Asiaticosides |
0 |
13.10± 0.11 |
26.34± 1.03 |
38.22± 1.07 |
47.89± 1.23 |
58.56± 0.32 |
|
|
F2 |
Curcumin |
0 |
6.0± 0.42 |
14.1± 0.10 |
34.2± 0.56 |
42.2± 0.66 |
47.18± 0.43 |
|
Asiaticosides |
0 |
11.34± 0.11 |
23.23± 1.03 |
36.20± 1.07 |
49.65± 1.23 |
54.30± 0.32 |
|
|
F3 |
Curcumin |
0 |
16.89± 0.10 |
37.11± 0.43 |
47.5± 0.29 |
56.23± 0.22 |
62.55± 0.76 |
|
Asiaticosides
|
0 |
19.34± 0.11 |
27.23± 1.03 |
40.20± 1.07 |
55.65± 1.23 |
69,12± 0.32 |
|
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
All three batches of transdermal patch (F1, F2, F3) shown significant amount of drug release. Among all three batches, the better drug release was observed in F3 formulation.
i) Invitro drug efficacy studies
The therapeutic efficiency of prepared herbal transdermal patch was analyzed by using invitro assays of free radical scavenging activity by DPPH and invitro anti-inflammatory assay since free radicals and chronic inflammation are one of the well identified causative factors for varicose vein.
a) Antioxidant activity by DPPH assay
The outcomes of invitro anti-oxidant activity are as follows:
Table 15. Invitro antioxidant study outcomes
|
Sr. No |
Name of Sample |
Conc. (µg/ml) |
% Inhibition |
|
1 |
Ascorbic acid (Reference standard) |
10 |
84.66±0.11 |
|
20 |
86.80±0.02 |
||
|
30 |
88.05±0.14 |
||
|
40 |
91.30±0.23 |
||
|
50 |
94.00±0.43 |
||
|
2. |
batch F1 |
10 |
60.75±0.23 |
|
20 |
63.32±0.11 |
||
|
30 |
65.90±0.34 |
||
|
40 |
70.82±0.22 |
||
|
50 |
71.77±0.56 |
||
|
|
batch F2 |
10 |
49.70±0.44 |
|
20 |
52.74±0.56 |
||
|
30 |
56.21±0.43 |
||
|
40 |
61.23±0.98 |
||
|
50 |
62.21±0.67 |
||
|
|
batch F3 |
10 |
64.48±0.33 |
|
20 |
67.95±0.76 |
||
|
30 |
73.09±0.55 |
||
|
40 |
78.02±0.87 |
||
|
50 |
87.62±0.30 |
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
|
|
|
|
Figure.5 Anti-oxidant activity curve of standard drug Gallic acid |
Figure.6 Anti-oxidant activity curve of F1 transdermal patch formulation |
|
|
|
|
Figure.7 Anti-oxidant activity curve of F2 transdermal patch formulation |
Figure.8 Anti-oxidant activity curve of F3 transdermal patch formulation |
In this study, all formulations F1, F2, F3 have shown free radical scavenging activity however, F3 batch shown significantly more anti-oxidant activity than F1 and F2. This may be due to the more drug release through combined polymer matrix.
b) Invitro anti-inflammatory assay
Chronic inflammation is one of the leading cause of development of varicose veins. Hence, it was crucial to analyze formulation for its therapeutic efficacy as an anti-inflammatory agent. The outcomes of this study are as follows:
Table 16. Anti-inflammatory activity outcomes of herbal transdermal patches
|
Sr. No |
Name of Sample |
Conc. (µg/ml) |
% Inhibition |
|
1 |
F1 |
12.5 |
37.29±0.02 |
|
25 |
40.30 ±0.04 |
||
|
50 |
37.88±0.23 |
||
|
100 |
40.84±0.11 |
||
|
2 |
F2 |
12.5 |
34.45±0.23 |
|
25 |
43.40 ±0.44 |
||
|
50 |
46.91±0.20 |
||
|
100 |
51.13±0.75 |
||
|
3 |
F3 |
12.5 |
40.73±0.34 |
|
25 |
43.20±0.55 |
||
|
50 |
50.90±0.32 |
||
|
100 |
61.03±0.16 |
Data expressed in terms of mean±SD. The data found to be statistically significant with p < 0>
In this study, all three batches of transdermal patch shown significant anti-inflammatory activity. However, batch F3 shown significantly more anti-inflammatory activity as compared to F2 and F3.
h) Stability Study
Stability studies were conducted in Environmental test chamber to assess stability of formulation with respect to their physical appearance, after storing them at 450c/75% RH for 3 months. The formulation found to be stable after 3 months period. No changes in physical appearance observed.
CONCLUSION
In conclusion, the herbal transdermal patch formulation F3 showcased potential as effective therapeutic agent for management of varicose vein. Ayurvedic herbs Centella asiatica and Curcuma longa whose chief active constituents are curcumin and asiaticosides, are poorly lipid soluble in their pure form. Hence incorporating them in transdermal patch can enhance their threrapeutic effect by enhancing skin permeation. In this way, developed herbal transdermal patch can be an effective strategy for varicose veins treatment. However, these outcomes needs to be confirmed by invivo studies before commercialization.
REFERENCES
Nameera Sadiya Begum, Anjali Kide-Nnadedkar*, Sudhakar Muvvala, Preparation And Evaluation of Herbal Transdermal Patch for Management of Varicose Vein, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 12, 1900-1913. https://doi.org/10.5281/zenodo.14451161
10.5281/zenodo.14451161
